Changes made on "mr-2021-22" "Non-classical disproportionation revealed by photo-CIDNP NMR"
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Changes made on „mr-2021-22“
“Non-classical disproportionation revealed by photo-CIDNP NMR”
The following changes have been made based on the comments of the two anonymous reviewers.
The changes refer to the line numbers in the manuscript with “Track changes” turned on!
mr-2021-22-rc1:
(1) “Research on photolyases and cryptochromes is presently a hot field. Cryptochromes are even
considered to be involved in “animal detected magnetic resonance”. Therefore, an update on flavin-
related compounds, their capacity to form radical pairs and to build up hyperpolarization is highly
desirable. The manuscript revealed the existence of a transient high redox state of 6,7,8-
trimethyllumazine by means of photo-CIDNP NMR. The compound is able to form upon illumination
radical pairs with its two different protonation states. The experimental isotropic hyperfine coupling
constants are obtained and correlate well with DFT data. Combining DFT and CIDNP, the careful
analysis of the data on the basis of Kaptein’s sign rule is convincing and allows a remarkable
reconstruction of a reaction dynamics involving photochemistry, spin-chemistry as well as proton
transfer.”
(2) We appreciate that this reviewer considers our contribution topical and the analysis well carried
out. Thank you!
(3) –
(1) “As for the title, I feel that the authors are unnecessarily opening a discussion that is not really
helpful. Why should a disproportionation (2A = A+ + A–) not also be able to be triggered
photochemically (A* + A = A+ + A–)? In the textbook by Klessinger and Michel, photochemically
triggered disproportionations are indeed discussed (for example, as a result of Norrish I). One could
also ask whether a system A + AH really “disproportionates”, since A and AH are different species.
Nevertheless, it seems to me that a very special CIDNP-capable donor-acceptor system is present here,
perhaps deserving the term “non-classical”.”
(2) In the suggested textbook by Klessinger and Michel we could not find any example of a
disproportionation that takes place directly following light excitation and promotion into an excited
multiplet state (excited singlet or triplet). The examples listed therein have disproportionation steps at
later stages of the overall reaction and are typically irreversible. The reviewer is correct in stating that
A (= TML–) and AH (=TMLH) in 6,7,8-trimethyllumazine (TML) are different species, but they are
protonation forms of the same molecular entity and in aqueous solution they are obligatorily
coexistent. Whether there are other cases of photo-disproportionations of the type in 6,7,8-
trimethyllumazine is very difficult (if not impossible) to find out. At least, there is no example we are
aware of. Therefore, we consider the photoinduced disproportionation of 6,7,8-trimethyllumazine as
“non-classical”, but could also accept the terms “non-standard” or “atypical” instead.
(3) see line 359. Unless the editor insists, we would leave the title as is.
This reviewer asks two questions:
(1) “From the Figure 6B, it seems like the H(7α) also enjoys very high amplitude of electron spin
density, but in the photo-CIDNP spectrum, there is no polarization at H(7a). How to understand such
a contradiction?“
1(2) The methyl group at position 7 is acidic with a pK value not far from that of the ammonium ion –
which is quite unusual. In D2O all three protons are replaced with deuterons and consequently there
are no proton resonances observed both in NMR and in photo-CIDNP, see line 89–91 of the original
manuscript. We will emphasize this proton-to-deuteron exchange more explicitly in a revised version.
(3) see lines 126 and 127.
(1) “In the supplementary information, Table S2, Table S3, there are three times H(6α), three times
H(7α), and three times H(8α). What do they refer to?“
(2) These are the individual hyperfine couplings of the three methyl protons per methyl group in the
rigid molecule. Their isotropic hyperfine values are averaged (due to fast methyl rotation on the NMR
timescale in aqueous solution and at room temperature) to yield the isotropic hyperfine values listed in
Table 1 of the main text.
(3) no text changes required.
This reviewer has also minor comments:
(1) “The “R.” of “Rhodobacter sphaeroides” needs to be introduced (line 35).“
(2) This will be corrected in a revised version. The same will be done with Agrobacterium (A.)
tumefaciens.
(3) see lines 35 and 36.
(1) ““it absorbs at shorter wavelengths” (line 41): Should an antenna not exactly do that? Might be
helpful to mention the absorption maxima.“
(2) This depends on the type of antenna and the wavelength range that shall be harvested. Why
cryptochromes harbor 6,7-dimethyl-8-ribityllumazine as antenna, which has a blue-shifted absorption
maximum (lmax = 420 nm) with respect to the flavin cofactor (lmax around 450 nm), cannot be
explained straightforwardly in light of the fact that the flavin itself absorbs significantly near 420 nm
and also shows an absorption maximum around 380 nm. Absorbance of an antenna could also be red-
shifted as long as there is overlapping absorbance with the moiety eventually “receiving” the
excitation energy.
The absorption maxima will be mentioned in the text in a revised version of the manuscript.
(3) see lines 41 and 42.
(1) ““operating at 14.1 T and a 1H freq of 600 MHz” (line 66): Sounds to be two different things.”
(2) This will be reworded in a revised version of the manuscript.
(3) see line 70.
(1) ““fibre” (line 70) = fiber.“
(2) This will be corrected in a revised version of the manuscript.
(3) see line 73.
(1) “Fig Caption Fig 3: Dashed lines show laser excitation.“
2(2) The caption of Fig. 3 will be supplemented accordingly.
(3) See caption of Fig. 3.
(1) “Line 212: radical pair formation from a triplet precursor: Is that surprising considering flavin
photo-chemistry?“
(2) One might expect a triplet precursor when comparing 6,7,8-trimethyllumazine to a flavin.
However, 6,7,8-trimethyllumazine has one aromatic ring less than flavin. Hence, the expectation of a
triplet pathway is in our opinion not obvious. Besides, there is at least one example of a flavin
performing electron transfer from its excited singlet state: the BLUF (blue light using flavin) domain.
(3) no text changes required.
(1) “Line 244: To what refers the “this”?“
(2) This sentence will be reworded in a revised version of the manuscript.
(3) see line 303 and 304.
(1) “Line 253: The use of the asterisk in a text of photochemical relevance might be misleading.“
(2) We will choose a different symbol for indicating hyperpolarization.
(3) see line 311.
mr-2021-22-rc2:
(1) “The manuscript “Non-classical disproportionation revealed by photo-CIDNP NMR” by Weber
and co-authors describes the results obtained by photo-CIDNP in the disproportionation reaction of the
compound 3, called 6,7,8-trimethyllumazine in its triplet state with 6,7,8-trimethyllumazine in the
ground state. The authors applied the pulsed variant of CIDNP detection with multiple repetition of a
short laser pulse followed by an RF pulse for NMR detection.
In 2011 direct proportionality between individual amplitudes of geminate CIDNP and the
corresponding hyperfine coupling constants in a multinuclear radical pair at high magnetic field was
theoretically predicted by the brilliant scientist Konstantin Ivanov, who too early passed away on
05.03 2021. The linearity was explained and verified in the publication of Morozova, O. B.; Ivanov,
K. L.; Kiryutin, A. S.; Sagdeev, R. Z.; Köchling, T.; Vieth, H.-M.; Yurkovskaya, A. V., Time-resolved
CIDNP: an NMR way to determine the EPR parameters of elusive radicals. Phys. Chem. Chem. Phys.
2011, 13 (14), 6619–6627. Prior to that, the Adrian model (F. J. Adrian, J. Chem. Phys., 1971, 54,
3918–3923) with a rather clumsy sum over all multiple nuclear spin projections was widely used for
CIDNP simulation. Please, refer to these publications in the manuscript.“
(2) These two publications will of course be mentioned in a revised version of the manuscript:
“In the high-field approximation, the applied time-resolved photo-CIDNP scheme using a pulsed laser
as light source (Closs et al., 1985; Goez 135 et al., 2005; Kuhn, 2013) renders signal intensities that
are proportional to the isotropic hyperfine coupling constant Aiso of the respective nucleus (Adrian,
1971; Morozova et al., 2011).” and “To rationalize our findings, we correlated the relative intensities
of the hyperpolarized NMR resonances obtained by photo-CIDNP to DFT predictions of hyperfine
couplings of the various paramagnetic one-electron oxidized or reduced lumazine species involved in
the suggested disproportionation scheme along a procedure introduced by Ivanov and Yurkovskaya
(Morozova et al., 2011), see Table 1, Fig. 5 and Supplementary Material.”
3(3) see lines 154 and 241.
(1) “The results obtained are very interesting and the manuscript is written quite nicely. However, I
agree with the comment of referee 1, that the general reaction scheme is needed to clarify the chemical
processes in aqueous solution leading to CIDNP formation.
For the special issue dedicated to Prof. Robert Kaptein who remarkably contributed to CIDNP as a
mature science, we decided to assist the authors of this nice manuscript and propose such a reaction
scheme to show the process resulting in CIDNP formation. The scheme is shown below. In a reaction
involving e-transfer coupled with proton transfer (PCET) between 6,7,8- trimethyllumazine in its
triplet state and 6,7,8-trimethyllumazine in the ground state the primary triplet radical pair of
(TMLHred•– TMLox•) is formed. It can either recombine or reversibly form (TMLH2red• TMLox•) via
protonation-deprotonation as shown in the scheme. On a second reaction path the radical pair
disproportionates with formation of the same products (the arrow indicating the release of a proton (–
H+), but with a different CIDNP pattern.“
(2) We appreciate the suggestion by this reviewer and have modified the general reaction scheme
accordingly, see new Figure 4:
Figure 4: Photoinduced reversible disproportionation of 6,7,8-trimethyllumazine. ISC, intersystem crossing.
(3) see new Figure 4 and its caption.
(1) “The resulting CIDNP pattern detected in the diamagnetic product of these reversible reactions is
formed at least in two pairs. For each pair, the own proportionality relationship between HFCCs and
CIDNP holds. For a similar situation, in the reaction of carboxy benzophenones, it was proposed to
utilize a linear combination of proportionality relationships to find the share of several radical pairs
that contribute to the CIDNP signal. (Morozova, O. B.; Panov, M. S.; Fishman, N. N.; Yurkovskaya,
A. V., Electron transfer vs proton-coupled electron transfer as the mechanism of reaction between
4amino acids and triplet-excited benzophenones revealed by time-resolved CIDNP. Phys. Chem. Chem.
Phys. 2018, 20 (32), 21127-21135). This procedure works very nicely for other cases as well (see
examples in the recent paper “Molecular features toward high photo- CIDNP hyperpolariztion
explored through the oxidocyclization of tryptophan” Phys. Chem. Chem. Phys., 2021, Advance
Article https://doi.org/10.1039/D0CP06068B).”
(2) We will reference these two publications.
(3) see line 294 and 295.
(1) “When applied to the reaction of the methylated lumazine the result is shown in figure 1 by the red
line and red symbols. Indeed, the linear combination of two radical pairs with shares of 0.23 and 0.77
provides perfect linearity with R2=1.“
(2) When writing this manuscript, we were considering a linear combination of two radical pairs
exactly as suggested by this reviewer. We did not dare to include such an analysis in the submitted
version because hyperfine predictions by DFT (as used here for the correlation to photo-CIDNP
intensities) are in many cases not “exact”: Computed hyperfine couplings depend on the used
functional, basis set and molecular geometry. Therefore, the weights of TMLH2red•(H(5)) and
TMLHred•– should be treated with caution as the choice of other parameters for DFT computation
might result in different hyperfine values. Nevertheless, the suggestion by this reviewer prompted us
to be bolder so that a quantitative analysis based on two different radical pairs will be included. Figure
5 will be modified accordingly.
Figure 5: Correlations of relative photo-CIDNP intensities to predicted Aiso values from DFT. TMLox• (blue line and filled
squares): slope = 0.0672 MHz–1, R2 = 0.9996; TMLHred•– (orange dotted line and open circles): slope = 0.0277 MHz–1, R2 =
0.7827; TMLH2red•(H(5)) (orange dashed line and filled circles): slope = 0.0248 MHz–1, R2 = 0.9863; linear combination of
TMLHred•– and TMLH2red•(H(5)) with a ratio of 0.228:0.772 (orange drawn line and half-filled circles): slope = 0.0262 MHz–
1
, R2 = 1. CIDNP intensities were multiplied by the respective sgn(Dg).
(3) See new Figure 5 and its caption as well as lines 290–298.
(1) „Moreover, the correct approach to proportionality of CIDNP vs HFCCs is to use one common
normalization of CIDNP intensities for the whole CIDNP spectrum, but not different ones for
individual patterns from different protonation forms as it is done in Table 2, and to combine
5proportionalities in a single plot (see Fig. 1). In this case, different slopes (that did not receive an
explanation by the authors) are clearly seen making it easier to follow the corresponding discussion.“
(2) We will change Figure 5 accordingly, see above. Explanations of the different slopes were given in
the original text, see lines 241 to 254.
(3) see Table 1.
(1) „Such a way of presenting the experimental data makes the interpretation of CIDNP data very
straightforward.“
“Since the interconversion case by protonation and deprotonation for two radical pairs is revealed and
explained in full accordance with the classical CIDNP theory proposed by Robert Kaptein, I strongly
recommend to remove the word “non-classical” from the title or rephrase it.“
(2) The word “non-classical” does not refer to the applicable CIDNP theory but to the type of
disproportionation as is manifested in 6,7,8-trimethyllumazine photochemistry. As mentioned in the
response to reviewer 1 (mr-2021-22-rc1), we could also accept the terms “non-standard” or “atypical”
instead of “non-classical”.
(3) Unless the editor insists, we would leave the title as is.
(1) „The CIDNP application to the studied reactions once again demonstrates the predictive power of
CIDNP introduced by Prof. Robert Kaptein whose 80th anniversary we all celebrate!
The paper can be accepted after a revision as described above.“
(2) We thank also this reviewer for the many valuable suggestions that helped improve this
contribution.
(3) no changes required.
Careful reading of the manuscript by a Ph.D. student from the University of Leipzig and by ourselves
revealed some additional typos, grammatical mistakes and inaccuracies, and missing information,
which we corrected resp. added, see
o line 52 (misspelling corrected),
o line 87 (missing a added),
o lines 123 and 124 (additional information on the number of scans provided),
o lines 175–177 (additional information on the starting geometry provided).
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